Integrative recombination of Lactobacillus delbrueckii bacteriophage mv4: functional analysis of the reaction and structure of the attP site

The integrase of the temperate bacteriophage mv4 catalyzes site-specific recombination between the phage attP site and the attB site of the host during lysogenization of Lactobacillus delbrueckii subsp. bulgaricus. The mv4 integrase also functions in a wide variety of gram-positive bacteria and in Escherichia coli. In this report, in vitro and in vivo recombination assays were developed and the integrase was purified in order to study in greater detail the mv4 attP × attB recombination event. In a cell-free extract of E. coli at 42° C, the mv4 integrase promotes efficient in vitro recombination between a supercoiled attP-containing plasmid and a linear attB fragment. The integrase, which was purified to apparent homogeneity, showed no absolute requirement for accessory factors, unlike the majority of the lambda Int family of recombinases. Deletion derivatives of the attP site were constructed and tested for recombination with the attB site in vitro. A 234-bp DNA fragment containing five scattered putative mv4 Int-binding sites was sufficient for function of the attP site. In contrast to the right arm of attP, most of the left arm could be deleted without drastically reducing the recombination efficiency. In vivo in E. coli, mv4 Int catalyzed recombination in trans between attP and attB sites present on two separate plasmids. This property was used to confirm in vivo the results of the deletion analysis of the attP site performed in vitro. Received: 22 July 1998 / Accepted: 4 June 1999     

A Universal Scaffold for Synthesis of the Fe(CN)2(CO) Moiety of [NiFe] Hydrogenase

Hydrogen-cycling [NiFe] hydrogenases harbor a dinuclear catalytic center composed of nickel and iron ions, which are coordinated by four cysteine residues. Three unusual diatomic ligands in the form of two cyanides (CN⁻) and one carbon monoxide (CO) are bound to the iron and apparently account for the complexity of the cofactor assembly process, which involves the function of at least six auxiliary proteins, designated HypA, -B, -C, -D, -E, and -F. It has been demonstrated previously that the HypC, -D, -E, and -F proteins participate in cyanide synthesis and transfer. Here, we show by infrared spectroscopic analysis that the purified HypCD complexes from Ralstonia eutropha and Escherichia coli carry in addition to both cyanides the CO ligand. We present experimental evidence that in vivo the attachment of the CN⁻ ligands is a prerequisite for subsequent CO binding. With the aid of genetic engineering and subsequent mutant analysis, the functional role of conserved cysteine residues in HypD from R. eutropha was investigated. Our results demonstrate that the HypCD complex serves as a scaffold for the assembly of the Fe(CN)₂(CO) entity of [NiFe] hydrogenase.     

Identification and characterization of a defective SSV1 genome integrated into a tRNA gene in the archaebacterium Sulfolobus sp. B12

Summary Within the chromosome of the archaebacterium Sulfolobus sp. B12, a 7.4 kb region was identified which displayed extensive sequence similarities to the 15.5 kb genetic element SSV1 carried by the same strain both as a circular form and as a site-specifically integrated copy. DNA sequence analysis indicated that this 7.4 kb region (designated SSV1^intB) represented an SSV1-like element distinguishable from the full-length integrated copy (designated SSV1^intA) by extensive deletions and point mutations. The physical organization of DNA sequences of SSV1^intB indicated that this element was integrated at the same attP site as previously identified for SSV1^intA. A comparison of the DNA sequences at the left attachment sites of SSV1^intA and SSV1^intB revealed that they both represented very similar putative arginine tRNA genes followed by a 10 by inverted repeat sequence. S1 nuclease mapping experiments indicated that these tRNA genes are transcribed. Offprint requests to: W. Zillig     

CHO细胞基因组中稳定hot spot位点研究进展

近些年来,治疗性重组蛋白类药物是生物制药领域研究的热点。工业化生产中常用于重组蛋白表达的细胞系是中国仓鼠卵巢(Chinese hamster ovary,CHO)细胞。传统CHO细胞系的表达大多数基于随机整合的方式,这可能会使目标基因整合到异染色质区域或者不稳定的染色质区域,导致CHO细胞表达不稳定,需要多轮筛选才能获得理想的表达细胞系。最新研究表明,外源基因在CHO细胞预测/特定的基因组位点中进行特异性整合,可以使重组CHO细胞的表达保持长期一致性和稳定性。CHO细胞基因组中高效稳定的转录整合位点被称为热点(hot spot)。阐述CHO细胞基因组稳定的hot spot位点近几年的研究进展,其中包括热门的hot spot位点,以及如何研究新的hot spot位点的方法。总结如何将外源基因高效定位于预测的CHO细胞hot spot位点,实现高水平稳定的表达重组蛋白,为发现新的有效的hot spot位点,构建稳定表达CHO细胞系提供参考。     

High biological activity of a recombinant protein immobilized onto polystyrene

The adsorption characteristics of glutathione S-transferases (GST) genetically fused with polystyrene (PS)-binding peptides (PS-tags) on PS plates with increase in hydrophilicity were studied to clarify the mechanisms of the specific interaction between the PS-tag-fused protein and PS plates. GST fused with the PS-tag PS19 (RAFIASRRIKRP) preferentially interacted with hydrophilic PS plates, even in the presence of high concentrations of competitors such as Tween 20 and BSA. Both basic and aliphatic amino acids in the PS-tags were involved in the specific interaction of PS-tags with the surface of the hydrophilic PS plate. Genetic fusion of the PS19 variants, PS19-4 (RAIARRIRR) and PS19-6 (RIIIRRIRR), further improved the immobilization yield of GST in the presence of a high concentration of the competitor BSA (50 mg/mL). The PS19-6 peptide specifically interacted with the surfaces of various hydrophilic PS plates, especially in the presence of Tween 20. Higher remaining activity was detected on all of the hydrophilic PS plates immobilized with GST-PS19-6 in comparison with those with wild-type GST and GST-PS19, and the remaining activity was further increased by the addition of Tween 20 in the adsorption state. The PS19-6 peptide developed in this study is therefore very useful as an affinity tag that can immobilize a target protein directly onto various hydrophilic PS supports with high remaining activity.     

Molecular Mechanism by Which Surface Antigen HP0197 Mediates Host Cell Attachment in the Pathogenic Bacteria Streptococcus suis

Streptococcus suis, one of the most important and prevalent pathogens inswine, presents a major challenge to global public health. HP0197 is an S.suis surface antigen that was previously identified by immunoproteomics and canbind to the host cell surface. Here, we investigated the interaction between HP0197 andthe host cell surface glycosaminoglycans (GAGs) using indirect immunofluorescence and celladhesion inhibition assays. In addition, we determined that a novel 18-kDa domain in theN-terminal region of HP0197 functions as the GAG-binding domain. We then solved thethree-dimensional structures of the N-terminal 18-kDa and C-terminal G5 domains usingx-ray crystallography. Based on this structural information, the GAG-binding sites inHP0197 were predicted and subsequently verified using site-directed mutagenesis andindirect immunofluorescence. The results indicate that the positively charged residues onthe exposed surface of the 18-kDa domain, which are primarily lysines, likely play acritical role in the HP0197-heparin interaction that mediates bacterium-host celladhesion. Understanding this molecular mechanism may provide a basis for the developmentof effective drugs and therapeutic strategies for treating streptococcal infections.     

Modulation of gain-of-function α6*-nicotinic acetylcholine receptor by β3 subunits

We previously have shown that β3 subunits either eliminate (e.g. for all-human (h) or all-mouse (m) α6β4β3-nAChR) or potentiate (e.g. for hybrid mα6hβ4hβ3- or mα6mβ4hβ3-nAChR containing subunits from different species) function of α6*-nAChR expressed in Xenopus oocytes, and that nAChR hα6 subunit residues Asn-143 and Met-145 in N-terminal domain loop E are important for dominant-negative effects of nAChR hβ3 subunits on hα6*-nAChR function. Here, we tested the hypothesis that these effects of β3 subunits would be preserved even if nAChR α6 subunits harbored gain-of-function, leucine- or valine-to-serine mutations at 9' or 13' positions (L9'S or V13'S) in their second transmembrane domains, yielding receptors with heightened functional activity and more amenable to assessment of effects of β3 subunit incorporation. However, coexpression with β3 subunits potentiates rather than suppresses function of all-human, all-mouse, or hybrid α6((L9'S or V13'S))β4*- or α6(N143D+M145V)(L9'S)β2*-nAChR. This contrasts with the lack of consistent function when α6((L9'S or V13'S)) and β2 subunits are expressed alone or in the presence of wild-type β3 subunits. These results provide evidence that gain-of-function hα6hβ2*-nAChR (i.e. hα6(N143D+M145V)(L9'S)hβ2hβ3 nAChR) could be produced in vitro. These studies also indicate that nAChR β3 subunits can be assembly partners in functional α6*-nAChR and that 9' or 13' mutations in the nAChR α6 subunit second transmembrane domain can act as gain-of-function and/or reporter mutations. Moreover, our findings suggest that β3 subunit coexpression promotes function of α6*-nAChR.     

利用苏云金芽胞杆菌转座子Tn4430构建含cry1Ac10基因的解离载体

将cry1Ac10基因和苏云金芽胞杆菌的复制起始区连 接在一起,并在其两侧按相同方向各连接一个来自苏云金芽胞杆菌转座子Tn4430的解离位点,构成转移单位。再将革兰氏阳性细菌的抗性标记基因和大肠杆菌克隆载体pUC19与之连接,获得含cry1Ac10基因的解离载体pBMB801。将其转化苏云金芽胞杆菌无晶体突变体,再导入含解离酶基因的辅助质粒pBMB1200。在解离酶作用下,两个解离位点间发生重组,消除基在操作中的抗性标记基因等非必需基因片段,获得仅保留有完整cry1Ac10基因和来 自苏云金芽胞杆菌质粒复制起始区,在无抗生素选择压力下能稳定遗传的重组质粒pBMB801B 。